Engineering DNA Delivery Polymers using Combinatorial and Cheminformatics Methods

描述(申请人提供)：拟议研究的总体目标是使用化学酶单体合成、平行聚合物合成和化学信息学建模来设计和评估用于转基因输送的聚合物。基于基因的策略，旨在通过引入外源基因来操纵细胞表型，在治疗和功能基因组学应用中具有吸引力。目前可用的非病毒(如聚合)基因传递载体受到毒性的限制，且效率较低。我们假设，合理的单体化学酶合成和组合聚合物合成的协同组合将导致具有高效向细胞传递基因的快速鉴定聚合物(特定目标1)。转基因数据的快速生成将有助于构建预测的定量结构-活性关系(QSAR)化学信息学模型，该模型将基因传递效率与聚合物和多链物化性质(例如，分子量、疏水性、Zeta电位等)联系起来。使用支持向量机(SVM)和核偏最小二乘(K-PLS)回归(具体目标2)。将开发一种新的QSAR模型-模型的方法，在该方法中，将首先使用基于单体结构的预测QSAR模型来估计聚合物的物理化学性质。然后，将使用这些估计的性质来生成转基因表达的QSAR模型，因此，对转基因表达有效性的预测将直接基于单体结构。从长远来看，这种预测性的QSAR模型将有助于合理设计高效的聚合物转染剂，这是一种有效的非病毒基因传递途径。认识到聚合物介导的转基因表达效率低下，我们将采用一种联合治疗方法，使用细胞内转运和转录(化疗增强剂)的介体，旨在增强细胞内的转基因表达(特定目标3)。最后，有效的聚合物和化疗增强剂将被用于传递编码TRAIL的基因，这种基因选择性地在体外和体内诱导癌细胞凋亡(特定目标4)。将采用SCID小鼠异种移植模型来研究22Rv1前列腺癌的消退，并将研究聚合物-质粒复合体的生物分布和毒性。预计拟议的研究将导致识别(1)用于非病毒基因传递的新的有效聚合物，(2)对影响转基因传递的聚合物物理化学因素的洞察，(3)将促进高通量有效聚合物识别的预测性QSAR模型，以及(4)基于聚合物传递的体内有效性和生物分布评估。预计这项研究将对基于基因的治疗、功能基因组学以及依赖于细胞中高水平转基因表达的各种应用产生重大影响。这项拟议的研究将开发用于基因传递的新型非病毒(聚合物)材料，并在体外和体内对其进行评估。它们将在许多疾病的基因治疗中得到重大应用，并将扩大这些病例的治疗选择。

DESCRIPTION (provided by applicant): The overall goal of the proposed research is to employ chemoenzymatic monomer synthesis, parallel polymer synthesis and cheminformatic modeling for the design and evaluation of polymers for transgene delivery. Gene-based strategies, designed to manipulate cellular phenotype by introducing exogenous genes, are attractive in therapeutic and functional genomics applications. Currently available non-viral (e.g. polymeric) gene delivery vectors are limited by toxicities and suffer from low efficacies. We hypothesize that a synergistic combination of rational chemoenzymatic synthesis of monomers and combinatorial polymer synthesis will lead to the rapid identification polymers with high efficacies for delivering genes to cells (Specific Aim 1). The rapid generation of transfection data will facilitate the construction of predictive Quantitative Structure-Activity Relationship (QSAR) cheminformatic models that correlate gene delivery efficacy with polymer and polyplex physicochemical properties (e.g. molecular weight, hydrophobicity, zeta potential, etc.) using Support Vector Machine (SVM) and Kernel-Partial Least Squares (K-PLS) regression (Specific Aim 2). A novel QSAR 'model- of models' approach will be developed in which, polymer physicochemical properties will first be estimated using predictive QSAR models based on monomer structure. QSAR models for transgene expression will then be generated using these estimated properties as a result of which, predictions of transgene expression efficacy will be based directly on monomer structures. In the long-term, such predictive QSAR models will aid in the rational design of high-efficacy polymeric transfection agents, which a powerful approach for non-viral gene delivery. Recognizing that polymer-mediated transgene expression suffers from low efficacies, we will employ a combination treatment approach using mediators of intracellular trafficking and transcription (chemotherapeutic enhancers), designed to enhance transgene expression in cells (Specific Aim 3). Finally, effective polymers along with chemotherapeutic enhancers will be employed for delivering genes that encode TRAIL, which selectively induces apoptosis in cancer cells, both in vitro and in vivo (Specific Aim 4). SCID mouse xenograft models will be employed to investigate recession of 22Rv1 prostate tumors, and biodistribution and toxicity of the polymer-plasmid complexes will be investigated. It is anticipated that the proposed research will result in the identification of (1) new effective polymers for non-viral gene delivery, (2) insights into polymer physicochemical factors that influence transgene delivery, (3) predictive QSAR models that will facilitate high-throughput 'in silico' identification of effective polymers, and (4) in vivo efficacy and biodistribution evaluation of polymer-based delivery. It is anticipated that this research will significantly impact gene-based therapeutics, functional genomics, and various applications that depend on high levels of transgene expression in cells. The proposed research will develop novel non-viral (polymeric) materials for gene delivery and evaluate them in vitro and in vivo. These will find significant application in gene therapy for a number of diseases and will expand therapeutic options in these cases.